This research program aims to obtain quantitative information of electrostatic interactions and dynamics in proteins. Significant new opportunities result for the availability of a cDNA clone for human myoglobin (mb) and an efficient system for producing the protein in E. coli. Part of the proposed research concerns structural and functional characterization of this important human protein. A fundamental question is the mechanism by which diatomic ligands enter and exit the iron binding site. Since there are no channels in the static X-ray structure, fluctuations are required for ligand access. This is a case where protein dynamics and biological function are directly related. Specific proposals of protein residues whose dynamics are crucial for ligand access are being tested systematically by site-specific mutagenesis. Ligand binding dynamics are measured from the femptosecond to kilisecond timescales by a wide range of spectroscopic techniques. Inherent in studies using site- specific mutagenesis is a bias that certain residues are more important than others. In order to avoid this bias a random mutagenesis strategy is outlined along with an approach to mass screening of mutants on the basis of ligand recombination kinetics. A second major area of research is to obtain quantitative information of electrostatic interactions in proteins using Mb mutants as a working model. Buried, potentially charged or polar amino acids are inserted in the sequence at defined locations. Information on the polarity of the protein interior and its effect on inter-residue electrostatic interactions is obtained by quantitative analysis of spectral and pKa shifts and changes in redox thermodynamics. A novel combination of non- photochemical holeburning and Stark effect spectroscopy is outlined to obtain information on the distribution of electrostatic fields in the heme pocket and the contribution of individual amino acid residues to the total electrostatic field. An approach to obtaining information of the time-dependent response of the protein interior to a sudden change in polarity is outlined. This is an important aspect of understanding the role played by the protein dielectric in regulating reactions such as electron transfer. An extension of earlier work in Mb and in homogeneous solution is proposed which involves measurement of the time-dependence of the emission spectrum of probe molecules in the heme pocket. Advances in ultra-fast fluorescence methods, theories of liquid dynamics and the possibility to examine the role of individual amino acid residues by site-specific mutagenesis make this possible.